Summary of work: One theory on the mechanism of aging of post-mitotic tissues proposes that an accumulation of oxidative damage to mitochondrial DNA (mtDNA) leads to impaired biogenesis of mitochondria and thus a failure to maintain cellular energy homeostasis. We are testing several elements of this hypothesis. In this project, we have quantitated the occurrence of two important oxidative DNA lesions, 8-OHdeoxyguanosine (8OHdG)and 8-hydroxy adenine in DNA using HPLC and LC/MSMS technique. In rat liver we found that 8-OHdG is more prevalent in mtDNA than in nuclear DNA. Further, there is a significant increase with aging; from 8 per 100000 deoxyguanosine residues at 6 months to 14 at 12 months and 22 at 23 months of age. There was no such age-linked change in nuclear DNA. We are measuring 5-OHcytosine as another marker for oxidative base damage of the mtDNA. We are also using another approach in which we can directly detect the presence of 8-OHdG in mtDNA in vivo with specific antibodies, thus minimizing the contribution of artificial oxidation during sample handling. These studies have been extended to mouse knockout models where we are able to determine whether specific DNA repair genes play a role in the persistence of oxidative DNA lesions in mitochondrial DNA. In particular, we have examined the OGG1 knockout mouse, defective in the major glycosylase that recognized 8OHdG in DNA. This mouse has a significant accumulation of 8OHdG in its nuclear DNA, but a much more dramatic increase in the 8OHdG levels in mitochondrial DNA. This leads to the conclusion that the OGG1 glycosylase is essential in mitochondrial repair of 8OHdG and that the function of this enzyme is more important in mitochondria than in the nucleus. We are now investigating whether caloric restriction can modulate the accumulation of 8OHdG in the OGG1 null mouse, since this regiment has been shown to decrease oxidative stress and extend life span. We have studied the human premature aging disease, Cockayne syndrome and find that it is associated with a defect in the repair of oxidative DNA base lesions in both the nucleus and mitochondria. Thus, we plan to investigate whether these repair deficiencies are associated with accumulation of oxidative DNA damage. In stably transformed cell lines where the cockayne protein is missing or is defect, we can detect the accumulation of oxidative DNA base lesions using LC/MSMS